Proposed Research

Microbial oxidation of methane in anoxic sediments constitutes the
major sink of methane in modern oceanic environments and largely
minimizes the marine contribution of the powerful greenhouse gas
methane to the atmosphere. The microorganisms involved in this process,
however, remained unknown for decades. All attempts to cultivate
them in the laboratory have failed.

With this proposal, we will establish and apply a new approach to
identify the key players in microbial methane oxidation in oceanic
sediments without cultivation. Samples from the Guaymas Basin hydrothermal
vent sites, harboring abundant methanotrophic microbial communities,
will be screened for molecular markers that are associated with
anaerobic methanotrophy and result from community members whose
role in anaerobic methanotrophy is still poorly constrained. Molecular
markers include specific membrane lipids with diagnostic carbon
isotopic signatures and ribosomal RNA sequences, the standard molecules
to place unknown organisms in the evolutionary tree of life. Promising
samples will be studied by fluorescence in situ hybridization (FISH)
of ribosomal RNAs, specifically targeting important microbial participants
in anaerobic methanotrophy with unknown function. Labeled cell aggregates
will subsequently be analyzed for their stable carbon isotope composition
by secondary ion mass spectrometry (SIMS).

This project will underpin WHOI's present leadership in the biogeochemical
and microbiological exploration of the most significant methane
sink in the oceanic realm as well as in developing new isotopic
and molecular strategies for studying natural microbial ecosystems.

With this project, we will accomplish two goals: (1) A more complete
understanding of the microbial ecology and diversity of microbial
anaerobic methanotrophic communities in marine sediments; and (2),
establishment of a novel technique at WHOI to measure isotopic compositions
of uncultured microbes, utilizing the WHOI Cameca IMS 1270 ion microprobe
- an approach that has potential to revolutionize investigative
strategies in environmental microbiology and microbial ecology.
These objectives address two of the three goals identified by the
Ocean Life Institute, "Biodiversity in the Ocean" and "New Tools
for Ocean Biology". Moreover, this project is also intimately linked
to current and future research projects of the PI's. For example,
a synergetic link can be foreseen to the NASA Astrobiology Institute
Subsurface Biospheres (including Teske and Hinrichs as PIs), which
focuses on the exploration of unknown microbial communities in the
Earth's subsurface.

Progress Report

Research funded by this grant was very dependent on the IMS 1270 ion microprobe, which was heavily damaged by the October 22, 2002 fire in the NENIMF facility at WHOI. As a consequence, we are continuing this work under a no cost extension that extends until June 30, 2005.

As of March 2004, Graham Layne is fully available to this project again - now that the initial abatement/remediation of the NENIMF laboratory spaces and equipment reorders for the IMS 1270 are complete. Since then, he has assembled a series of carbon isotope standards specifically suited to the analysis of small objects such as bacterial consortia and microfossils, and is currently characterizing their d13C by conventional gas SIRA mass spectrometry.

We are planning to travel (Layne/Hinrichs) to the NORDSIMS IMS 1270 facility in Stockholm this fall to analyze a series of samples of pyritiferous carbonate nodules from methane-derived limestones forming around marine seeps. The samples are being supplied by Prof. J. Peckmann, Bremen University. We suspect that strong variations in the d34Spyrite and d13Ccarbonate contents from the center toward the periphery of the nodules will be observed - a consequence of the process of anaerobic oxidation of methane (AOM), which is responsible for the carbonate precipitation at these seeps.

This study of nodule samples will provide novel information on the carbon (and sulfur) isotope fingerprints of this particular microbial process. It will also allow us to establish our procedures for d13C analysis of microbial samples in this alternate facility. Subsequently, we will return to our study of d13C and trace element concentrations in microbial cultures that was underway at the time of the NENIMF fire, using the capability established in Stockholm.

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